PROJECT SUMMARY Over $200 billion dollars' worth of biological products, including antibodies, vaccines and proteins, are produced each year. This continues to grow worldwide at about 15%/year, making biopharmaceuticals a fairly recession-proof, growing and pro?table industry. This shift towards biopharmaceuticals re?ects a fundamental shift within the pharmaceutical industry. Biomanufacturers are under constant pressure to reduce costs by increasing productivity of their cell culturing activities. We believe, by taking advantage of post-transcriptional regulation, that we can achieve a 20%-50% improvement in productive yield, defined as titer, as well as significant reductions in workload and time. For perspective, a 25% improved titer from each cell in production could save $16 billion in biomanufacturing costs. Current biomanufacturing practice is to express a drug resistance gene as a selectable proxy for successfully transformed cells, which are then sub-cloned and screened for production of the GOI. These basic techniques have not changed in any substantial way in over 20 years, but they have two major drawbacks: (1) they require the production of the antibiotic resistant gene which competes with the GOI for scarce translational resources placing an additional metabolic burden on the cells, and (2) the selection marker or antibiotic resistance gene production is not directly coupled to the GOI giving the potential for false positives. Our protocol, PTSelect, uses post-transcriptional regulation as an alternative to using a drug resistance gene by using an siRNA coupled as an intron to the GOI. Rather than force the cells to transcribe and translate an additional drug resistance gene that serves as a proxy for GOI expression, we introduce a custom siRNA into an intron upstream of the GOI. Expression of the tethered siRNA and GOI is thus directly coupled making future selection potentially more accurate. We then use mRNA to perform selection with an mRNA has sequences that are perfectly complementary to the siRNA to induce RNA interference (RNAi) ultimately down regulating the death gene mRNA. Thus, the more the GOI is produced, the more the siRNA is also produced which results in more degradation of the mRNA. Instead of adding a chemical to select for resistance, we transfect a death gene encoding mRNA into the cells, or a fluorescent marker than identifies desired cells using fluorescence-activated cell sorting (FACS), or a cell surface marker gene mRNA coupled with magnetic-bead-Ab to perform magnetically activated cell sorting (MACS). This project will allow us to finished development of these techniques and performa a comparison study between current resistance gene products and our new PTSelect product.